Enhanced Growth and Drought Resistance in Seedlings of Acacia
tortilis due to Inoculation of Arbuscular Mycorrhiza Fungi and Bacillus
subtilis
Abdelmalik M Abdelmalik*, Thobayet S
Alshahrani and Abdulaziz A Al-Qarawi
Department of Plant production, Faculty of Food and
Agriculture Sciences, King Saud University, Saudi Arabia
*For correspondence: aadam1@ksu.edu.sa
Received 15 October 2021; Accepted 23 November 2021;
Published 15 December 2021
Abstract
A shade house experiment was conducted in Saudi Arabia
to evaluate the impact of a mixture of three arbuscular mycorrhiza fungi (AMF)
namely Funneliformis mosseae, Rhizophagus intraradices and
Claroideoglomus etunicatum, a bacterium Bacillus subtilis, and their
combinations on the growth and drought resistance potential of Acacia
torilis seedlings under moderate and water deficit-stress. Thus, inoculants
treatments (AMF, Bacillus subtilis, AMF+Bacillus, and control)
and several watering intervals (1, 2, 3 and 4 weeks) were applied. Inoculation
of AMF and Bacillus to A. tortilis seedlings
found effective in terms of improved seedling growth. AMF and combined
inoculation resulted in a larger shoot (shoot fresh and dry weights, seedling
height, leaf number, leaf area) and root development (root fresh and dry
weights, root length, root surface area, and root volume) as compared to the
non-inoculated seedlings. Single inoculants of B. subtilis, showed
better improvement in 1- and 2-week watering intervals compared to the control.
Inoculated seedlings showed lower proline accumulation than non-inoculated
seedlings, and thus improved seedling resistance to water deficit-stress.
Mycorrhizal and mixed inoculation enhanced the amount of chlorophyll in the seedling’s
leaves. Furthermore, seedlings with AMF and co-inoculants showed better drought
tolerance even at 3- and 4-week watering intervals. © 2021 Friends Science
Publishers
Keywords: Acacia tortilis; AMF; Bacillus
subtilis; Co-inoculation; Deficit-stress
Introduction
Drought and climate change are great challenges that is
being faced by forest ecosystems today (Bhuyan et al. 2017) and
prediction of climate change models suggest that drought risk will rise in
tropical forests during the next years. Results of different experiments state
that drought can cause a reduction in trees development and amplified trees
mortality (Richard 2016). Drought can cause significant environmental effects
and is likely to increase in many places in the world with climate change
(Amanda et al. 2016), particularly in the arid regions, where water is a
limiting factor that controls plant growth and survival (Kondoh et al.
2006).
A large number of plants make symbiotic relationship
with microorganisms in the soils to overcome the negative impacts of the
drought (Wang and Qui 2006; Nadeem et al. 2014). Rhizosphere
microorganisms have a decisive role on the growth of plants established under
limiting soil environments (Hashem et al. 2019). Microorganism
association provides essential resources to the plant, and that in turn will
improve the performance of plant to cope with drought (Liddycoat et al.
2009). Different eco-physiological studies have stated that AMF symbiosis is a
key factor that assists plants to cope with water stress and increase drought
tolerance (Javaid 2009; Rapparini and Penuelas 2014). Yooyongwech et al. (2013)
showed that AMF symbiosis enhanced chlorophyll content in woody trees under
water deficit conditions. The inoculation of plant growth promoting bacteria
(PGPB) singly or combined with other microorganisms (such as AMF) are
widespread and their application is rising in global farming practices
(Díaz-Zorita and Fernández-Canigia 2009; Sharf et al. 2021). The combination
of AMF fungi and Bacillus subtilis increased the fresh and dry biomass
production of aromatic plants (Alam et al. 2011). Hashem et al.
(2015) concluded that there is a positive effect of B. subtilis on the growth of inoculated plants. Also, they stated
that B. subtilis strain caused significant increase in chlorophyll a
and b content in leaves of Bassia indica.
Acacia tortilis is one of the
widespread tree across the dry-lands of African continent (especially Sudan)
and Middle East and has a great role for several groups of pastoral communities
(Andersen 2012). The tree is considered an important species of the arid region
in many African and Asian countries, where provides building wood, shade,
forage, shelter for people and animals, richness the biodiversity, and keep
soil fertile, so that it is considered a keystone species (Maarten et al.
2015; Verma 2016).
The effect of water deficit-stress on plant life is
affected by the plant growth period, length and strength of the water
deficit-stress (Sharma et al. 2020). However, tree seedling stage is the
most sensitive phase to the water deficit-stress for many plant species
(Arrieta and Suárez 2006), because of their limited root networks that mostly
found at the topsoil layer which make them experience further severe water
deficiency than large trees and eventually, drought can lead to its destruction
completely (Mueller et al. 2005). Seedling’s stage of A. tortilis life cycle is the most critical stage;
therefore, in this study we investigated the impact of co-inoculation of AMF
and B. subtilis on growth and tolerance of A. tortilis
seedlings to water deficit-stress.
Materials and Methods
Mycorrhizal
fungi
The mycorrhizal fungi in our study consisted of a
combination of Funneliformis mosseae (Syn. Glomus mosseae), Rhizophagus
intraradices (Syn. Glomus intraradices) and Claroideoglomus
etunicatum (Syn. Glomus etunicatum). These AMF fungi were extracted
and isolated from the hair roots of Conocarpus erectus trees. AMF
species were identified following the protocol defined by Redecker et al.
(2013), where spores were separated and observed under computerized compound
microscope. The identification process depended on the morphological
characteristics of the spores.
Propagation
of AMF
Inoculums of the mycorrhizal fungi were developed for 4
weeks in pots containing Sudan grass (Sorghum sudanense). The source
inoculums were taken from the fine roots of Conocarpus erectus trees at
the faculty of Agriculture and Food Sciences, King Saud University (KSU) and then placed
in autoclaved sandy soils. After that, seeds of the host plant (Sudan grass)
were spread in the pots. Pots were irrigated as needed until the host plant
(Sudan grass) grown and established and become ready to be applied as
inoculums.
Bacterium inoculants preparation
B. subtilis, was isolated previously from the roots of Acacia
seyal Benth trees (Alqarawi et al. 2014; Hashem et al. 2015).
The inoculants of B. subtilis were prepared in small flasks (250 mL), each flask has
100 mL of nutrient medium and then flasks were incubated on a shaker for three
days at 25°C. Afterwards, the bacillus suspensions were adjusted to 3.6 × 109
cfu mL−1.
Plant culture
and growth conditions
Seeds of A. tortilis were
provided by Forestry Research Centre, Khartoum, Soba, The Republic of the
Sudan. The experiment was carried out in the shade house, Faculty of Food and Agriculture
Sciences, KSU, from March to June. Seeds were sown in a plastic pot (50 cm
height and 16 cm diameter). Pots were filled by a sandy loamy soil (3:1 v/v),
with following characteristics: 0.42% of organic carbon with 0.075–0.10 mm
particle size. The procedures described by Sommers (1982) and Miller (1987) was
used for particle size and organic carbon analysis.
In each pot two seedlings were established in sterilized
sandy loam soil. Pots were inoculated with AMF, Bacillus, and
co-inoculants (AMF + Bacillus). Inoculation of AMF was done to the soil
before seeding process. For bacillus treatment, seeds were dipped in the B. subtilis suspensions
for 10 minutes and then talc powder was added as an adhesive material. After
that, seeds were removed from suspension and dried at room temperature and then
planted in the soil. Further suspension was added to the soil of Bacillus
and combination treatments to increase cell number of Bacillus in the seedling’s
rhizosphere. Another group of seedlings was established under the same
environment but without inoculants (control). Seedlings were irrigated
frequently until the second true leaf was shown, after that, the seedlings were
exposed to four irrigation intervals where pots irrigated by 250 mL of water
every 1, 2, 3 and 4- weeks watering interval (water-deficit treatments).
Design of the
experiment and layout
A split-plot arrangement in randomized complete block
design was used to set up the experiment. Treatment consisted of four drought
intervals (1, 2, 3 and 4 weeks) and four groups of microorganism’s treatments;
control (no microorganisms), AMF, B. subtilis and co-inoculants (AMF + Bacillus) with four
replications (pot) per treatment.
Root
colonization by mycorrhizal fungi
AMF were
extracted from root hairs samples of AMF and co-inoculants treatments following
the method defined by Daniels and Skipper (1982) and modified by Utobo et
al. (2011). The roots were well washed with distilled water to remove the
soil particles adherent to it, then washed with KOH (10%) and afterward stained
with trypan blue in lactophenol, as followed by Phillips and Hayman (1970). The
stained root hairs were cut to small segments, and then checked by a bright
microscope at 400 × 23 magnification. Mycorrhizal fungi infection (mycelium, vesicles
and arbuscules) in root hairs was measured using the following formula:
Spore extraction
AMF Spores were separated using wet sieving and
decanting method (Gerdemann and Nicolson 1963). A 100 g of soil samples were air dried and 800 mL of water was
added to generate soil suspension. The suspension was filtered using
gradual sieves. Then, suspension was filtered through gridded Whatman filter
paper No. 1. The filter paper was tested under microscope at 2.5 × 10
magnification and then spores number was recorded.
Measurements
of areal and root part traits
Roots fresh
weight, stems, and leaves were separated from each other and were weighed using
a digital balance scale. Then, the roots, vegetative part (stem and leaves)
were individually dried at 75°C for 48 h to achieve dry weights.
The following traits were measured during experiment: height
of plant (cm) from the cotyledon scars to the seedling apex using a ruler,
seedling stem diameter (mm) at the cotyledon scar using a digital caliper (± 0.04
mm), leaves number, leaf area by using portable leaf area meter (Model CI-202,
CID, Bio-Science, Camas, USA) and branches number.
Seedlings were smoothly taken out from the soil, and
then roots were separated from the shoot. Seedling roots were washed well from
the adhesive soil and then spread gently over a scanner device connected with a
computer and then and scanned at 600 dots per inch. The root images were saved
in TIFF format to be evaluated and measured by a computer software. The root
traits (total root length (cm), root surface area, root volume and root dimeter
were measured using WinRhizo Pro software (Regent Instruments Inc and Christian
1996).
Estimation of chlorophyll a and b
Fresh leaf
samples were collected from each treatment. Sample of fresh leaves with 0.5 g
per treatment was weighted using digital balance scale and then placed at glass
tube. Each tube filled with 5 mL of diemethyl formamide and left for 24 h at
room temperature. After 24 h, leaf extracts were filtered and placed in
spectrophotometer cuvette and read absorption at 664 nm, for chlorophyll a and
at 620 nm for Chlorophyll b (Porra et al. 1989).
Estimation of Proline accumulation
Sadasivam and
Manickam (1996) protocol was followed to measure seedlings proline content.
Where, small sample from seedlings leaves (0.5 g) were clipped in the early
morning and grounded in mortar and pestle by adding 10 mL of 3%
sulphosalicyclic acid and the resulted homogenate was centrifuged at 18000 g
for 1 h and purified. Then, 2 mL of filtered solution were added in test
containers to glacial acetic acid (2 mL) and acid ninhydrin (2 mL) and test
containers were watery bathed for 1 h at 100°C, followed by ice bath. The
reaction blend was vortexed with toluene (4 mL). Layer of toluene was separated,
and the absorbance was measured using spectrophotometer at 520 nm (Genesis
10-S, Thermo Fisher Scientific, Madison, USA). A standard curve of proline was
used to identify proline accumulation.
Statistical
analysis of the data
Analysis of variance (ANOVA) was used to analyze the
data and means were separated using Fisher’s least significance difference test
(LSD) at P < 0.05.
Statistical analyses were done using the SPSS software package version 22.0.
Results
Infection of A. tortilis
roots by AMF
AMF colonization rate: AMF obviously colonized the
roots of A. tortilis at mycorrhizal and
co-inoculants treatments (Fig. 1). The highest colonization rate was recorded
at co-inoculant treatments at all irrigation interval. The greatest
colonization percentage (93.3% for mycelium, 77.2% for vesicle, and 68.1% for arbuscular)
which was recorded at co-inoculant treatment at 1-week irrigation intervals. The
lowest colonization rate (50% for mycelium, 14.7% for vesicle, and 17.9% for
arbuscular) at 4, 2, 3-weeks irrigation interval respectively (Table 1).
Spores density: The spores’ total densities
varied between irrigation intervals and between AMF and co-inoculant treatment.
The highest number of spores was (104 spores 10 g-1) which recorded
at co-inoculant treatment, and at 1-week watering interval, however, the lowest
spore’s number was (30 spores 10 g-1) which found at the AMF
treatment at 4-weeks irrigation interval (Table 1).
Effect of inoculants on shoot fresh and dry weight of A.
tortilis seedlings
Results of statistical analysis showed significant impact
for inoculants treatments (co-inoculant, AMF, Bacillus) on shoot fresh
and dry weight in all irrigation intervals compared to control (Table 2). The
seedlings treated with co-inoculants showed the highest averages for shoot
fresh and dry weights in all irrigation intervals, followed by AMF-treated
seedlings, and then B. subtilis treated seedlings. On other hand, control seedlings
showed the lowest shoot fresh and dry weights (Table 2). In comparison to the
control seedlings, co-inoculant increased shoot fresh weight by (207.07,
495.44, 916.77 and 792.65%) and shoot dry weight by (177.43, 1482.97, 891.17
and 651.24%) at 1, 2, 3 and 4weeks irrigation intervals respectively. However,
AMF treated seedlings increased by 140.40, 1181.20, 659.02 and 626.95%, (for
shoot fresh weight) and 148.44, 980.69, 668.38 and 636.93%, (for shoot dry
weight), at 1, 2, 3 and 4-weeks irrigation interval. For B. subtilis treated
seedlings, the percentage of increments were 26.63, 736, 5.62 and 5.32% (for
shoot fresh weight) and 1.49, 511.02, 2.83 and 5.68% (for shoot dry weight), at
1, 2, 3 and 4-weeks irrigation interval.
Effect of inoculants on vegetative growth of A.
tortilis seedlings
Statistical
analysis indicated that co-inoculants treatment significantly affected
seedlings height, leaf number and leaf area of the seedlings, compared to the
control (Table 3). In the different irrigation interval, seedlings treated with
co- Table 1: Percentages of AMF
colonization and spore’s number in the roots and rhizosphere of A. tortilis seedlings
Irrigation interval (weeks) |
Inoculant treatment |
AMF Colonization rate (%) |
Spores’ numbers |
||
Mycelium |
Vesicle |
Arbuscular |
|||
1 |
AMF |
76.9 |
27.0 |
47.8 |
76 |
|
Co-inoculant |
93.3 |
77.2 |
68.1 |
104 |
2 |
AMF |
66.6 |
20.0 |
26.6 |
52 |
|
Co-inoculant |
74.1 |
14.7 |
55.2 |
60 |
3 |
AMF |
63.3 |
20.0 |
20.0 |
48 |
|
Co-inoculant |
85.5 |
25.8 |
17.9 |
46 |
4 |
AMF |
50.0 |
23.3 |
30.0 |
30 |
|
Co-inoculant |
67.3 |
14.8 |
27.6 |
36 |
Table 2: Effect of co-inoculant on shoot fresh and dry weights
of A. tortilis seedlings under different
irrigation intervals
Irrigation interval (weeks) |
Inoculant treatment |
Shoot fresh weight (g/plant) (Means ±SE) |
Shoot dry weight (g/plant) (Means ±SE) |
1 |
Control |
1.7900 ± 0.1127d |
0.8867 ± 0.1186d |
AMF |
4.3033 ± 0.2603b |
2.2033 ± 0.1090ab |
|
Bacillus |
2.2667 ± 0.2624c |
0.9000 ± 0.0473d |
|
Co-inoculant |
5.4967 ± 0.1849ab |
2.4600 ± 0.1350a |
|
2 |
Control |
0.2500 ± 0.0416e |
0.1533 ± 0.0145e |
AMF |
3.2033 ± 0.3569bc |
1.6567 ± 0.1617bc |
|
Bacillus |
2.0900 ± 0.2312c |
0.9367 ± 0.1004d |
|
Co-inoculant |
5.6567 ± 0.0982a |
2.4267 ± 0.1405a |
|
3 |
Control |
0.2367 ± 0.0133e |
0.1167 ± 0.0120e |
AMF |
1.7967 ± 0.2196d |
0.8967 ± 0.1213d |
|
Bacillus |
0.2500 ± 0.0470e |
0.1200 ± 0.0208e |
|
Co-inoculant |
2.4067 ± 0.3421c |
1.1567 ± 0.1172cd |
|
4 |
Control |
0.2233 ± 0.0353e |
0.1167 ± 0.0203e |
AMF |
1.6233 ± 0.1849d |
0.8600 ± 0.0924d |
|
Bacillus |
0.2633 ± 0.0524e |
0.1233 ± 0.0120e |
|
Co-inoculant |
1.9933 ± 0.3023d |
0.8767 ± 0.1091d |
|
LSD 0.05 |
|
1.2692 |
0.5691 |
Sig |
|
*** |
*** |
Mean
± standard errors. Values with same letters differ non-significantly at (P > 0.05)
Fig.
1: Infection and colonization of A. tortilis
roots by AMF
inoculants showed the greatest average of height which
was 45.33 cm, 49.66 cm, 60.00 cm, and 50.66 cm at 1, 2, 3 and 4-weeks
irrigation interval, respectively. The average of leaf number/plant was also
the greatest in the all-different irrigation intervals with average of 34.333
(at 1-week irrigation intervals), 36.33 (at 2-weeks irrigation interval), 41.66
(at 3-weeks irrigation interval), and 17.33 (at 4-weeks irrigation interval). Leaf
area also has the highest average in co-inoculants treatment, which was 92.667
cm2, 131.85 cm2, 37.663 cm2 and 37.043 cm2,
at 1, 2, 3 and 4-week irrigation intervals, respectively (Table 3).
Effect of inoculants on root growth of A. tortilis seedlings
Co-inoculants, AMF and Bacillus treatments,
significantly improved root fresh and dry weights in A. tortilis seedlings
under the different drought conditions. Both co-inoculants and AMF treated
seedlings showed the greatest average of shoot fresh and dry weights (Table 4).
At 3 and 4-week irrigation intervals, drought decreased root fresh and dry
weight of control and bacillus treated seedlings, however co-inoculated and
AMF-seedlings were not affected by drought in terms of root fresh and dry
weight (Table 4). Similarly, inoculum treatments had positive effect on the
root parameters (root length, root surface area, root volume, and root
diameter); where AMF and co-inoculants treatments showed the greatest root
system at all irrigation intervals (Table 5 and Fig. 2–5).
Effect of inoculants on chlorophyll a and b
Inoculant’s treatments significantly improved the
content of chlorophyll-a in the leave of A. tortilis
seedlings. No significant different was observed in chlorophyll b,
however, seedlings inoculated with co-inoculants, AMF, and B. Table 3: Effect of
co-inoculant on height, leaf number and leaf area of A. tortils
seedlings under different irrigation intervals
Irrigation Intervals (weeks) |
Inoculant treatment |
Height (cm) (Means ± SE) |
Leaf number (Means ± SE) |
Leaf area (cm2) (Means ± SE) |
Leaf temperature °C (Means ± SE) |
1 |
Control |
18.667 ± 4.6667fg |
13.667 ± 0.3333fg |
34.577 ± 2.1219ef |
29.933 ± 0.6936a |
AMF |
54.000 ± 1.7321a |
46.000 ± 1.5275a |
124.16 ± 3.0751a |
26.367 ± 1.6707abcd |
|
Bacillus |
29.667 ± 3.3830def |
21.667 ± 1.2019de |
50.253 ± 2.1040d |
25.067 ± 0.4910bcde |
|
Co-inoc |
45.333 ± 2.1858abc |
34.333 ± 1.2019c |
92.667 ± 3.1714b |
24.033 ± 1.2811cde |
|
2 |
Control |
21.667 ± 1.4530efg |
10.000 ± 0.5774gh |
25.827 ± 1.1526fg |
29.567 ± 0.3844ab |
AMF |
39.333 ± 2.8480bcd |
27.000 ± 0.5774d |
43.887 ± 1.2714de |
28.100 ± 0.8505abcd |
|
Bacillus |
32.333 ± 1.4530cde |
19.333 ± 0.6667ef |
38.133 ± 0.1764def |
23.833 ± 1.0525de |
|
Co-inoc |
49.667 ± 0.3333ab |
36.333 ± 0.3333bc |
131.85 ± 3.8031a |
21.633 ± 0.5840e |
|
3 |
Control |
12.000 ± 0.5774g |
5.0000 ± 0.5774h |
15.140 ± 0.1701gh |
30.667 ± 0.4256a |
AMF |
43.333 ± 0.3333ab |
36.667 ± 2.0276bc |
32.703 ± 2.7629ef |
29.267 ± 0.2963ab |
|
Bacillus |
14.667 ± 0.8819g |
6.0000 ± 0.5774h |
9.1400 ± 0.3100h |
27.700 ± 0.9539abcd |
|
Co-inoc |
54.667 ± 4.6667a |
41.667 ± 2.7285ab |
37.663 ± 1.6709def |
26.967 ± 0.2186abcd |
|
4 |
Control |
13.000 ± 0.5774g |
7.6667 ± 0.3333gh |
11.533 ± 0.7860h |
28.533 ± 1.3836abcd |
AMF |
39.000 ± 3.0551bcd |
18.333 ± 0.8819ef |
68.973 ± 4.6321c |
28.733 ± 0.2728abc |
|
Bacillus |
14.333 ± 0.6667 g |
10.000 ± 0.5774gh |
6.4400 ± 0.4903h |
28.733 ± 0.5696abc |
|
Co-inoc |
50.667 ± 1.8559 ab |
17.333 ± 0.8819ef |
37.043 ± 3.1542ef |
26.267 ± 0.2963abcd |
|
LSD 0.05 |
|
13.262 |
6.5313 |
12.986 |
4.8349 |
Sig |
|
*** |
*** |
*** |
* |
Mean ± standard
errors. Values with same letters differ non-significantly at (P > 0.05)
Table
4: Effect of
co-inoculant on root length, root surface area, and root tips number of A. tortils seedlings under different irrigation intervals
Irrigation interval (weeks) |
Inoculant treatment |
Root fresh weight (g) (Means ± SE) |
Root dry weight (g) (Means ± SE) |
1 |
Control |
2.7167 ± 0.2554bc |
1.6933 ± 0.0736bcd |
AMF |
4.3667 ± 0.4914ab |
2.2500 ± 0.2616ab |
|
Bacillus |
3.7567 ± 0.3779abc |
1.4633 ± 0.1027bcd |
|
Co-inoculation |
5.1167 ± 0.3805a |
2.2767 ± 0.1172ab |
|
2 |
Control |
0.1767 ± 0.0536d |
0.1133 ± 0.0120f |
AMF |
3.6100 ± 0.3444abc |
1.7633 ± 0.1387bcd |
|
Bacillus |
2.3167 ± 0.2293c |
1.0867 ± 0.0176cde |
|
Co-inoculation |
4.1300 ± 0.3208abc |
1.4833 ± 0.2350bcd |
|
3 |
Control |
0.1533 ± 0.0120d |
0.0800 ± 0.0115f |
AMF |
5.3167 ± 0.4667a |
2.7833 ± 0.2530a |
|
Bacillus |
0.2800 ± 0.0001d |
0.1700 ± 0.0115ef |
|
Co-inoculation |
3.1533 ± 0.5128bc |
1.7767 ± 0.2826bcd |
|
4 |
Control |
0.2267 ± 0.0338d |
0.1200 ± 0.0321f |
AMF |
2.3733 ± 0.3457c |
1.0033 ± 0.1586def |
|
Bacillus |
0.3233 ± 0.0273d |
0.2067 ± 0.0145ef |
|
Co-inoculation |
3.9267 ± 0.6274abc |
1.9867 ± 0.2765abc |
|
LSD 0.05 |
|
1.8283 |
0.9614 |
Sig |
|
*** |
*** |
Mean ± standard errors. Values with same letters differ
non-significantly at (P > 0.05)
subtilis showed better content of chlorophyll-b than control
seedlings (Table 6).
Effect of inoculants on proline accumulation
The proline accumulation in the leaves of all seedlings
improved by increasing the irrigation intervals (Table 7). However, seedlings
inoculated with co-inoculants, AMF, and bacillus showed lower content of
proline than in control seedlings regardless of the irrigation intervals. This
variation was more obvious under varied irrigation intervals.
Discussion
Responses of the plant to the soil's microbes are the
consequence of interaction relationship between plants and microbes found in
the soil. It obviously appears from our results that A. tortilis seedlings
much improved and resisted drought when AMF and their combination with B. subtilis were
applied.
Inoculation with AMF, Bacillus and co-inoculants
improved plant height, leaf number, leaf area, and shoot fresh and dry weights. This
improvement could be due to the increased presence of carbohydrates in the
shoot part, enhancement of nutrients uptake (Verma et al. 2018) and
increase of root system in treated seedlings. Our results in the same line with
many studies that found, AMF improves plant growth by increasing nutrients
amount in the soil and its absorption to the plant (Naheeda et al. 2020;
Ya-Dong et al. 2021).
Table 5: Effect of co-inoculant on root length, root surface
area, root volume and root diameter of A. tortils
seedlings under different irrigation intervals
Irrigation
interval (Weeks) |
Inoculant treatment |
Root length (cm) (Means ± SE) |
Root surface area(cm2) (Means ± SE) |
Root volume (Means ± SE) |
Root diameter (Means ± SE) |
1 |
Control |
414.47 ± 24.056b |
90.870 ± 6.9272bc |
1.8367 ± 0.1135ce |
0.7233 ± 0.0318bc |
AMF |
697.14 ± 4.9770a |
232.31 ± 34.631a |
3.5067 ± 0.2373ab |
1.1600 ± 0.0600a |
|
Bacillus |
554.28 ± 66.016ab |
168.39 ± 13.964ab |
3.1333 ± 0.1648ab |
0.7867 ± 0.0273bc |
|
Co-inoculation |
618.13 ± 76.139ab |
160.26 ± 8.8719ab |
3.6233 ± 0.5732ab |
0.7833 ± 0.0521bc |
|
2 |
Control |
152.39 ± 8.5729c |
34.137 ± 4.4081bc |
0.6933 ± 0.0751e |
0.7400 ± 0.0404bc |
AMF |
424.22 ± 31.882b |
197.06 ± 60.199ab |
3.2267 ± 0.3548ab |
0.7000 ± 0.0404bc |
|
Bacillus |
523.64 ± 83.834ab |
132.62 ± 18.121abc |
1.7533 ± 0.0120ce |
0.7500 ± 0.0643bc |
|
Co-inoculation |
348.25 ± 15.681bc |
140.03 ± 9.3029ab |
4.5767 ± 0.4421a |
1.1233 ± 0.0426a |
|
3 |
Control |
96.890 ± 6.5094c |
21.010 ± 1.1252c |
0.4167 ± 0.041e |
0.6400 ± 0.0764
bc |
AMF |
373.33 ± 25.584bc |
135.50 ± 3.0394abc |
4.3667 ± 0.1913a |
1.1633 ± 0.0940a |
|
Bacillus |
135.13 ± 12.871c |
39.097 ± 3.3340bc |
0.9033 ± 0.0736ce |
0.9233 ± 0.0233ab |
|
Co-inoculation |
418.36 ± 33.081b |
160.22 ± 18.291ab |
3.4700 ± 0.7410ab |
1.1433 ± 0.0581a |
|
4 |
Control |
152.38 ± 6.5865c |
55.617 ± 9.9760bc |
1.0233 ± 0.0433ce |
0.5867 ± 0.0176c |
AMF |
597.11 ± 43.652ab |
146.42 ± 14.684ab |
2.4000 ± 0.0651bc |
0.6267 ± 0.0463c |
|
Bacillus |
152.94 ± 22.496c |
31.597 ± 3.8153bc |
0.5200 ± 0.0557e |
0.6567 ± 0.0296bc |
|
Co-inoculation |
567.35 ± 57.092ab |
174.36 ± 7.8967ab |
4.0100 ± 0.2201a |
0.7700 ± 0.0100bc |
|
LSD 0.05 |
|
245.47 |
124.84 |
1.5887 |
0.2734 |
Sig |
|
*** |
** |
*** |
*** |
Mean ± standard errors. Values with same letters differ non-significantly at (P > 0.05)
Table 6: Effect of co-inoculant on chlorophyll-a, chlorophyll-b,
and proline accumulation of Acacia tortilis
seedlings under different irrigation intervals
Irrigation
interval (Weeks) |
Inoculant treatment |
Chlorophyll-a
(Means ± SE) |
Chlorophyll-b
(Means ± SE) |
1 |
Control |
1.9857 ± 0.0110
bc |
1.3440 ± 0.0832ab |
AMF |
2.9003 ± 0.0454a |
1.7140 ± 0.0188ab |
|
Bacillus |
2.9523 ± 0.0288
a |
1.6060 ± 0.2441ab |
|
Co-inoculation |
2.9477 ± 0.0268
a |
1.7217 ± 0.2319ab |
|
2 |
Control |
2.2950 ± 0.0277b |
1.2723 ± 0.0598b |
AMF |
2.3320 ± 0.0519
b |
1.4120 ± 0.1868ab |
|
Bacillus |
2.7450 ± 0.0279
ab |
1.1680 ± 0.2480b |
|
Co-inoculation |
2.6863 ± 0.2295
ab |
1.5350 ± 0.0594ab |
|
3 |
Control |
1.5680 ± 0.0150c |
1.2320 ± 0.0576b |
AMF |
1.9073 ± 0.0135bc |
1.6567 ± 0.0640ab |
|
Bacillus |
1.8723 ± 0.0306
bc |
1.1723 ± 0.0351b |
|
Co-inoculation |
2.4057 ± 0.0872ab |
1.5570 ± 0.0188ab |
|
4 |
Control |
1.4153 ± 0.2040
c |
1.2600 ± 0.1123b |
AMF |
1.5793 ± 0.0946
c |
1.4537 ± 0.2041ab |
|
Bacillus |
1.5987 ± 0.0345
c |
1.2657 ± 0.0198b |
|
Co-inoculation |
2.4500 ± 0.2306
ab |
2.0600 ± 0.0259a |
|
LSD 0.05 |
|
0.5655 |
0.7779 |
Sig |
|
*** |
NS |
Mean ±
standard errors. Values with same letters differ non-significantly at (P > 0.05): NS: not significant
Table 7: Effect of co-inoculant on proline accumulation of Acacia
tortilis seedlings under different irrigation
intervals
Irrigation
interval (Weeks) |
Inoculant
treatment |
Proline
content (Means ± SE) |
1 |
Control |
1.1013 ± 0.1366abc |
AMF |
0.7383 ± 0.0184bc |
|
Bacillus |
0.5027 ± 0.0256c |
|
Co-inoculation |
0.5490 ± 0.0142c |
|
2 |
Control |
1.0380 ± 0.1217abc |
AMF |
0.8843 ± 0.0616bc |
|
Bacillus |
0.7523 ± 0.1289bc |
|
Co-inoculation |
0.4223 ± 0.0225c |
|
3 |
Control |
1.3727 ± 0.0353ab |
AMF |
0.8570 ± 0.0749bc |
|
Bacillus |
1.2637 ± 0.3669abc |
|
Co-inoculation |
0.9873 ± 0.0552abc |
|
4 |
Control |
1.5873 ± 0.2492a |
AMF |
0.8850 ± 0.0111bc |
|
Bacillus |
0.8840 ± 0.0341bc |
|
Co-inoculation |
0.7677 ± 0.0528bc |
|
LSD 0.05 |
|
0.7255 |
Sig |
|
* |
Mean ±
standard errors. Values with same letters differ non-significantly at (P > 0.05): NS: not significant
Fig. 2: Effect of inoculants on root architecture of Acacia tortilis seedlings at 1-week irrigation intervals
A: Control treatment; B: Bacillus subtilis
treatment; C: AMF treatment; D: co-inoculation treatment
Fig. 3: Effect of inoculants on root architecture of Acacia tortilis seedlings at 2-weeks irrigation intervals
A: Control
treatment; B: Bacillus subtilis treatment; C: AMF treatment; D: co-inoculation
treatment
Fig. 4: Effect of inoculants on root architecture of Acacia tortilis seedlings at 3-weeks irrigation intervals
A: Control treatment; B: Bacillus subtilis
treatment; C: AMF treatment; D: co-inoculation treatment
Fig. 5: Effect of inoculants on root architecture of Acacia tortilis seedlings at 4-weeks irrigation intervals
A: Control
treatment; B: Bacillus subtilis treatment; C: AMF treatment; D:
co-inoculation treatment
with our
previous results in other acacia species (Abdelmalik et
al. 2020). Addition of B. subtilis strain (pf4) (Anand et al.
2010), to plant resulted in a significant vigor index, shoot height and root
length (Gowtham et al. 2020). Bacillus species can form endospores that
are extremely resilient to harsh environmental conditions and can also produce
metabolites that increase growth and vigor of plant. Also, many
exopolysaccharides can be produced by bacillus which help water uptake by plant
roots (Hashem et al. 2019). Zaidi et al. (2009) stated that B.
subtilis acts directly involved in the dissolution of phosphorous and plays
a synergistic role with the AMF. AMF alone increased the growth rate by 49.4%;
however, when combined with B. subtilis, growth rate increased by 59.5%
(Alam et al. 2011). The combined application of AMF and B.
subtilis has a synergistic role and
leads to promotion of plant growth (Hashem et al. 2019).
Results indicated to varied positive effect for AMF,
co-inoculant and B. subtilis, in the different irrigation intervals and
even at severe water-deficit conditions the inoculants showed positive effect
on seedlings growth. Microorganisms have tremendous capabilities to reduce
environmental stress and their interactions with plants, so that they offer
both a local and systemic defense under various environmental stresses (Chialva
and Bonfante 2018; Khoshru et al. 2020). Mycorrhizal fungi regulate and
improve plant growth when exposed to harsh environmental conditions, where they
significantly
enhanced the growth (Yadav et al. 2018; Xiao
et al. 2019; Abdelmalik et al.
2020; Yasser et al. 2021) and biomass of tobacco plants under normal
conditions and mitigated the decline caused by water deficit-stress (Begum et al. 2020). In this
regard, that inoculation of Onobrychisvicii folias seedlings with AMF
reduces the damage resulted from water deficit-stress and improved the water
deficit-stress resistance up to forty days (Kong et al. 2014).
Interactions between AMF and plant growth promoting rhizobacteria (PGPR) in the
plant rhizosphere has a synergistic role which improve growth and quality of
the plant (Khalid et al. 2017). PGPR can greatly enhance plant growth
and show beneficial interaction between plant and microbes. B. subtilis
enhances stress tolerance in plants by stimulating the expression of stress
response genes, hormones and metabolites related with drought stress (Lee et
al. 2014; Hashem et al. 2019).
AMF, co-inoculants, and B. subtilis treated
seedlings showed greater root length, root surface area, root diameter, and
root volume than non-treated seedlings at all irrigation intervals, which would
enable inoculated plants to explore great volume of rhizosphere and hence more
nutrients availability to the seedlings. It clear that, the effects of AMF,
bacillus, and co-inoculants on root morphology might be an important reason for
enhanced nutrients uptake for the treated seedlings. Our results agree with a
number of studies which reported that mycorrhizal fungi alters root morphology
and increases plant tolerance to the severe environmental conditions (Khanna et al. 2019). Largest root morphology result in
better nutrient uptake (Ya-Dong et al. 2021) and enhanced water
relationships in the plants (Pallavi and Sharma 2021). The mixed inoculants (B.
subtilis + AMF) improved root biomass and plant survival rate in comparison
to those caused by sole inoculations and non-inoculated plants (Ibrahim et
al. 2019). The combined use of PGPR may have a synergic effect on
decreasing contrasting stress factors. The application of PGPR with useful
fungi in farming is a suitable use in some stressful conditions (Deepmala et
al. 2019; Hassan and Bernard 2020). Occurrence of PGPR is highly linked
with plant rhizosphere and positive direct and indirect impacts on plant
development; like a decline in environmental stress is reported. Bacillus
species can make endospores that are tremendously resilient to severe
environmental conditions and also can produce metabolites that motivate plant
development and fitness (Hashem et al. 2019).
Inoculum’s treatments were found to have significant
contribution in the improvement of chlorophyll-a and b in A. tortilis seedlings under different irrigation
intervals. The improvement of chlorophyll content in AMF and co-inoculated
seedlings can be justified by the availability of nutrients and metabolism in
the plant. However, AMF improve nutritional status of plants by absorbing and
translocating mineral nutrients beyond rhizospheric zone (Rouphael et al.
2015). Microorganisms were found to improve the content of plant chlorophyll
under normal and water deficit-stress conditions. Co-inoculated plants have
higher chlorophyll content compared to single inoculants of AMF or bacteria
(Mehdi et al. 2018). Also, the findings reported by Kim et al.
(2010) and Berta et al. (2014) proved that co-inoculation (AMF and
bacteria) increased the levels of chlorophyll content in plants leaves. The
positive effect of AMF was extensively reported by scientists. Various research
results explained that the association of microorganisms to the plant can
change its physiological growth under many stress conditions (Xiaoying et
al. 2014). Mycorrhizal inoculation highly improved the content of
chlorophyll-a, b, and total chlorophyll (Naheeda et al. 2020) in the Erythrina
variegata leaves. Yooyongwech et al. (2013) and Fang et al.
(2018), showed that AMF symbiosis under water deficit conditions enhances
chlorophyll fluorescence in woody tree nut species. Sonal et al. (2018)
found that total chlorophyll content was more in AMF maize seedlings when
compared to non-treated plants where AMF- maize seedlings had double of
chlorophyll content as compared to control maize seedlings. AMF colonization
could promote the synthesis of chlorophyll and carotenoid thereby enhancing the
photosynthesis and biomass accumulation in plants through increasing the root
absorption area and root activity, support the absorption and transport of
water and other nutrients or mineral elements such as P, K, Mg, and Mn (Baslam et al. 2013). Also, in mycorrhizal plants the
increase of chlorophyll contents can be associated with increased P and Mg
uptake (Zhu et al. 2014).
Proline concentration increased greatly in the leaves of water deficit-stressed and non-inoculated
seedlings compared to the well-watered and inoculated seedlings. The lower
proline content in AMF and co-inoculation seedlings is an indicator of good
drought tolerance of plant (Ruiz-Lozano 2003), therefore, the low content of
proline in the inoculated seedlings in this study was linked with good
seedlings drought tolerance that is induced by AMF and co-inoculant treatments.
This finding is in agreement with Yooyongwech et al. (2013), where they
stated that, AMF and co-inoculation in different plant species reduces proline
content when water level is limited. The work
done by Doubkova et al. (2013), explained
that, when the concentration of proline increases in response to drought
stress, a lower proline accumulation has been observed in AMF- plants. In the
same way, Wu and Xia (2006) reported that, the content of proline was reduced
significantly in orange seedlings inoculated by AMF under water stress
conditions. The study conducted by Hazzoumi et al. (2015) reported that
leaf proline accumulation was greater in non-AMF plants than AMF-plants under
water deficit-stress. The synergistic effects between the bacillus and AMF were
reported to increase nutritional status of inoculated plants and thus stimulate
the plants resistance to the water deficit-stress (Ibrahim et al. 2019).
The changes made by PGPR on root elasticity are one of the essential steps to
enhanced tolerance to water shortage (Dimkpa et al. 2009). PGPR enhances
the plant cell membranes by stimulating the antioxidant system and increasing
drought tolerance of many plant species (Gusain et al. 2015).
Furthermore, AMF plants mostly had better leaf water status and high root
volume, thus, plants suffer less water deficit and consequently had lower
proline accumulation. The lower proline accumulation in the AMF plants may
derive from the integration of the inhibition of glutamate synthetic pathway of
proline with an enhancement of proline degradation (Zou et al. 2013).
Conclusion
Several soil microorganisms positively affect the growth
and drought tolerance of A. tortilis seedlings,
especially in the early stages of their growth, even when exposed to severe
water deficit-stresses. The addition of inoculants in general and co-inoculants
and AMF in particular resulted in increases in vegetative growth rates (fresh
and dry weight, height, number of leaves, leaf area) and root traits (fresh and
dry weight, root length, root surface area, root volume). In addition, the
inoculants led to an improvement in some physiological characteristics such as
chlorophyll-a and b. Also, inoculums reduced proline concentration levels in
water deficit-exposed seedlings, and therefore improved seedlings drought
tolerance and reduced damage resulting from water deficit due to more water
content in inoculated plants. It can be said that, changes in proline levels
are a response to tolerance or avoidance for water deficit. Based on the
attained results, the inoculants can be used for A. tortilis seedlings,
especially during the establishment stage. This will assist in the success of
afforestation programs in the dry areas.
Acknowledgments
The authors thank the Deanship of Scientific Research,
King Saud University, Saudi Arabia for supporting this work.
Author Contributions
AM, T S, and AA planed the experiment.
AM conducted the experiment, data measurements and analysis, and wrote the
first draft. TS analyzed root data and supervised all work. AA supervised the
work.
Conflicts of Interest
All authors declare no conflicts of
interest
Data Availability
Data presented in this study will be
available on a fair request to the corresponding author
Ethics Approval
Not applicable in this paper
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